There is an increasing interest in recombinant Gly-Xaa-Yaa-proteins, (wherein Gly is glycine and Xaa and Yaa are independently any amino acid) such as gelatins. The widespread use and possibilities for use of these Gly-Xaa-Yaa proteins in medical and clinical applications means that economically viable production processes are required. This has prompted careful consideration of process variables and variations in protein sequences that could influence expression properties, and hence yields of the desired Gly-Xaa-Yaa-proteins.
Naturally occurring Gly-Xaa-Yaa-proteins such as gelatins, which are obtained by hydrolyzing collagen derived from animal tissues, are actually mixtures of a very large number of peptides and polypeptides. The molecular weight range of these (poly)peptides depends on the tissue from which the gelatin was derived and the processing conditions.
In principle, the recombinant production of Gly-Xaa-Yaa-proteins yields products in which all the component molecules have the same length (the same number of amino acid residues), since a particular gene typically encodes a single protein (barring genes that contain introns). The coding sequence of the gene determines the length of the nascent polypeptide. Secretory proteins also typically contain a signal sequence (signal peptide, preprotein sequence) that is removed during secretion. The remaining polypeptide is then considered to be the mature polypeptide.
Gly-Xaa-Yaa-proteins are sensitive to various proteases. Thus, Gly-Xaa-Yaa-proteins that are secreted by commonly used expression hosts such as Pichia pastoris or Hansenula polymorpha are heterogeneous in size due to (partial) digestion by intracellular and extracellular proteases.
In a strict interpretation only the mature full length polypeptide is considered to be the product. However in the field of repetitive biopolymers often both the mature polypeptide and its proteolytic fragments as considered the final product.
In the first interpretation, proteolysis can lead to a significant reduction in the yield of the product and the degradation products may pose a significant problem during purification of the mature protein (because of the similarity between the mature protein and its degradation products). The product in the sense of the second interpretation has some similarity to animal-derived gelatins: a product that comprises polypeptides with various lengths. However, the heterogeneity of this product can be disadvantageous in some applications.
Therefore, prevention of proteolysis will benefit both the yield and the quality of the product. Proteolysis can be avoided by inactivation of particular protease genes or by excluding target sites for these proteases in the product. Examples of both approaches can be found in the literature. EP926543 and Werten et al. 1999 (Yeast 15, 1087-1096) describe a production method of Gly-Xaa-Yaa proteins using the methylotrophic yeast Pichia pastoris, where the Gly-Xaa-Yaa proteins corresponding to fragments of the helical domains (consisting of Gly-Xaa-Yaa triplet repeats) of mouse type I collagen (encoding a 21 kDa and 28 kDa, calculated MW, COL1A1 peptide and a 53 kDa COL1A2) and rat type III collagen (COL3A1) are produced. A factor such as the fermentation pH was in certain cases found to influence the stability of the expressed product. The presence in the expressed sequence of target sites for certain proteases was also thought to be relevant.
In US 2006/0241032 XRGD-enriched gelatin-like proteins with a minimum (increased) level of XRGD motifs and with a certain distribution of said XRGD motifs are disclosed that were found to be highly suitable for cell adhesion and cell binding in medical and biotechnological applications. The cell binding peptides described therein have good cell attachment properties. However, susceptibility to degradation has been a limiting factor in the ability to produce large amounts of specific Gly-Xaa-Yaa-proteins. In EP2112997 XRGD-enriched gelatin proteins are disclosed that have an improved stability. This was achieved through avoidance of the use of particular amino acid residues (Asp, Pro, Hyp) as the X in the XRGD motifs in the sequence. However further improvement of the stability of recombinantly produced gelatin-like Gly-Xaa-Yaa proteins is desired.
The proteases in most expression systems are not known. Even less information is available on the targets of these proteases and their substrate specificity. Therefore, in most cases, sequences that are resistant to proteolytic attack must be determined empirically. In the current patent application we disclose new Gly-Xaa-Yaa-proteins that can be produced with improved yields and quality by the exclusion of proteolytic target sites that have not been previously identified